Haematology, Biochemistry and Morphological Features of Peripheral Blood Cells in Captive Boa Constrictor - PMC

20/10/23, 9:47 Haematology, biochemistry and morphological features of peripheral blood cells in captive Boa constrictor - PMC
As a library, NLM provides access to scientific literature. Inclusion in an NLM database does
not imply endorsement of, or agreement with, the contents by NLM or the National Institutes
of Health.
Learn more: PMC Disclaimer | PMC Copyright Notice
Conserv Physiol. 2023; 11(1): coad001. PMCID: PMC9885740

Published online 2023 Jan 28. doi: 10.1093/conphys/coad001 PMID: 36726862
Haematology, biochemistry and morphological features of peripheral blood cells in

captive Boa constrictor
E Dervas, E Michalopoulou, A Liesegang, M Novacco, F Schwarzenberger, U Hetzel, and A Kipar
Steven Cooke, Editor
Assessment of health and disease in reptiles is challenging as basic health parameters are not
known for many reptile species. We undertook an in-depth study to determine basic blood pa‐
rameters, stress hormone levels and the morphological features of peripheral blood cells in
Boa constrictor, a common captive reptile species.
Abstract
The common boa (Boa constrictor) belongs to the family Boidae and represents one of the
most popular traded and kept snake species in captivity. The early diagnosis, prevention and
prophylaxis of diseases in this species, and in reptiles in general, still pose major challenges,
also due to the lack of reliable reference values. This prompted us to conduct a study on clini‐
cally healthy captive B. constrictor to assess their basic health parameters in the blood (haema‐
tological and biochemical values, stress markers). Several parameters differed significantly be‐
tween younger (<3 years) and older (≥3 years) boas; in the latter, the percentages of
eosinophils, the haemoglobin and haematocrit levels, as well as the albumin and total protein
levels, were higher. In male snakes, cholesterol levels were significantly higher than in females.
Light and electron microscopy as well as immunohistochemistry served to identify and deter‐
mine the morphological features of peripheral blood cells, that is, heterophils, basophils,
eosinophils, azurophils, monocytes, lymphocytes, thrombocytes and erythrocytes. Leukocyte
subpopulations, that is, T and B cells and monocytes, were also identified based on specific
marker expression. The study provides data on haematological, biochemical and stress hor‐
mone levels, suitable as reference values, and on the blood cell morphology of B. constrictor
which can serve as a guideline for further research on this species.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9885740/ 1/33
Introduction
Boa constrictor, the sole species historically allocated to the monotypic genus Boa of the family
Boidae, is a large non-venomous viviparous snake indigenous in tropical forests of Central and
South America. The B. constrictor is one of the longest-lived snakes with a life span in captivity
of 20 years or more (Divers, 1996). It is one of the snake species most frequently traded as
well as kept and bred in captivity (Montgomery et al., 2015). In most countries, the husbandry
of B. constrictor follows defined guidelines that, despite being closely adapted to the natural en‐
vironment, subject the animal to very ‘standardized’ housing conditions regarding environ‐
mental temperature and humidity, enclosure size etc., with only little seasonal variation (van
Waeyenberge et al., 2018). Captivity in general has often been stated to expose reptiles to
stress, favouring the development of immunosuppression and, therefore, infectious diseases
(Warwick et al., 2013; van Waeyenberge et al., 2018; Morgan and Tromborg, 2007;
Schumacher, 2006). Another risk for infections comes from the mixing of species from differ‐
ent geographic regions, resulting in the potential exposure to pathogens that the animals have
previously not been in contact with (Schumacher, 2006). B. constrictor are susceptible to a wide
range of viral, bacterial and parasitic infections. Important viral agents reported in this species
are reptarenaviruses (the causative agents of Boid inclusion body disease, BIBD) (Hetzel et al.,
2013) and the ophidian paramyxovirus (Piewbang et al. 2021). Parasitic infections are also of
relevance; reported are, for example, arthropods (mites, e.g. Ophionyssus natricis), intestinal
parasites (e.g. Cryptosporidia) and blood parasites (Hepatozoon sp.) (García-Má rquez et al.,
2019).
Although knowledge on appropriate maintenance and upbringing of captive snakes is growing,

the early diagnosis, prevention and prophylaxis of infectious diseases still poses major chal‐
lenges. In general, establishing a definitive clinical diagnosis is often difficult in reptiles, as the
animals show only few pathognomonic clinical signs and often have a chronic disease course
(Divers, 1996). In both human and veterinary medicine, haematological and biochemical pa‐
rameters are important, widely used markers for assessment of the health status, clinical diag‐
nosis and prognosis of disease, and to monitor the response to treatment. They are also funda‐
mental tools to monitor the health status of wild and captive reptiles (Christopher et al., 1999;
Lisičić et al., 2013). However, in contrast to domestic animals, haematological and biochemical
data in reptiles are currently not as readily available because reference values are still lacking
for most species (Nardini et al., 2013). The latter may also be the reason why haematologic and
biochemical values in reptiles are often interpreted in analogy to those of mammals (Grego et
al., 2006). Establishment of haematological reference intervals is also hardened by the incon‐
sistent classification of leukocyte subpopulations in reptiles, as some authors characterized pe‐
ripheral blood cells based on their function (Carvalho et al., 2017), others solely based on their
morphology (Svoboda et al., 2006; Zimmerman et al., 2010).
Similarly, the determination of stress in reptiles, particularly of chronic stress, also remains
challenging. Corticosterone levels are commonly used as biomarker for stress in reptiles (van
Waeyenberge et al., 2018), yet the interpretation of the results is difficult as, similar to blood
parameters, they are strongly dependent on several extrinsic parameters (eg. temperature,
season, habitat, diet, disease, stress, venipuncture site) as well as intrinsic factors (species, sex,
age, physiologic status) (Joseph, 2015).
Studies that provide reference values for haematological, biochemical and stress markers in
boid snakes are sparse and focus mainly on wild animals and their seasonal variations
(Fonseca Sarmiento et al., 2018; Machado et al., 2006; Quadrini et al., 2018). The present study
aimed to establish basic haematologic and biochemical parameters and to gather data on the
‘stress level’ of clinically healthy captive B. constrictor, to obtain a diagnostic tool set for the as‐
sessment of their health status.
Material and Methods
Animals. The study was undertaken on blood samples collected from 79 clinically healthy B.
constrictor originating from collections of Swiss (A, C-L) and German (M) breeders or shelters.
Twenty-one animals (B1-B21) originated from a reptile shelter that takes over reptiles from
private owners or breeders who are unable to maintain their collection. The snakes were euth‐
anized as the shelter had not succeeded in finding new owners for the animals within a pro‐
longed period of time. Euthanasia was performed at the Institute of Veterinary Pathology,
Vetsuisse Faculty, University of Zurich, followed by blood collection and a full diagnostic post
mortem examination to exclude any disease and to obtain information on the general health
status in the facility. Information on the exact origin of the snakes was not available.
In all animals, the blood sampling was undertaken upon the owners’ request and with their full
consent, with the aim to diagnose or exclude BIBD and reptarenavirus infection, as a means to
determine the infection status of the shelter and the collections, respectively. In live animals
(A10-A28, C1-C7, D1-2), it was performed onsite in the collection. All blood sampling was un‐
dertaken on the basis of a project permit (cantonal number ZH 195/2016 and ZH136/2020)
from the Cantonal Veterinary Office in Zurich.
In the cases where euthanasia was performed, an ASPA (Animals Scientific Procedures Act
1986) schedule 1 (appropriate methods of humane killing
(http://www.legislation.gov.uk/ukpga/1986/14/schedule/1)) procedure was applied.
All animals were tested negative for BIBD by cytological examination of a blood smear (see
below).
Blood sampling
Blood samples were collected from living animals by cardiocentesis using a 22- or 25-gauge
needle on a 3-ml syringe. The heart was visualized by a SonoTrax Vascular Doppler (FS15575,
EDAN, USA). A blood volume of 1–2 ml was collected into heparinized tubes (Sarstedt,
Germany), the volume depending on the size of each animal but not exceeding 5–8% of the to‐
tal blood volume (Nardini et al., 2013).
From dead animals, blood was extracted by cardiocentesis using a 22- or 25-gauge needle on a
3-ml syringe immediately after euthanasia.
Blood cytology
Smears were prepared from all blood samples and stained with May–Grü nwald–Giemsa. They
were subjected to a cytological examination to identify the morphological features of leukocyte
subpopulations and to determine the BIBD-negative status of the animals (no evidence of the
pathognomonic cytoplasmic inclusion bodies (IB) within blood cells), as previously described
(Hetzel et al., 2013).
Buffy coat preparation, processing and staining, immunohistochemistry, RNA-ISH and

transmission electron microscopy
In 13 animals, the blood volume was sufficient to also prepare a buffy coat. Plain micro haema‐
tocrit capillary tubes (BR749321, Merck, Germany) were filled with blood and spun in a micro‐
haemofuge (Heraeus Medical AG, Switzerland) at 12 700 g for 5 min according to previously
described protocols (Fontes Pinto et al., 2018). Tubes were broken at the plasma-buffy coat in‐
terface using a diamond pen, and the remaining portion of the capillary tubes (containing the
buffy coat) immersed in either 4% formalin for 24 h for light microscopy, or 2.5% glutaralde‐
hyde for 24 h and then buffered in 0.2 M cacodylic acid buffer, pH 7.3 for transmission electron
microscopy (TEM).
The formalin-fixed buffy coat pellets were paraffin wax embedded. Consecutive sections (3–
5 μm) were prepared, stained with haematoxylin eosin (HE) and subjected to RNA in situ-hy‐
bridization (RNA-ISH) and immunohistochemical staining, respectively.
Immunohistochemistry served for the identification of T cells (CD3+) and cells with monocytic
morphology (monocytes and azurophils; Iba1+), applying cross-reacting antibodies and the
horseradish peroxidase method, and using a Dako autostainer (Dako, Glostrup, Denmark).
Briefly, after deparaffinization, antigen retrieval was performed by incubation of the slides with
citrate buffer (pH 6) for Iba1 or EDTA buffer (pH 9) for CD3 at 98°C for 20 min in a pressure
cooker. After incubation with the primary antibodies, rabbit anti-human Iba1 (1:350, 019–19
741, Wako, Osaka, Japan) overnight at 4°C and mouse anti-human CD3 (1:150, M725401, clone
F7.2.38, Dako) for 1 h at room temperature (RT), sections were incubated with the secondary
antibodies (EnVision+ HRP Rabbit (Dako) for anti-Iba1, EnVision+HRP Mouse (Dako) for anti-
CD3) according to the manufacturer’s protocol. Endogenous peroxidase was blocked by incu‐
bation with peroxidase blocking solution (Dako) for 10 min at RT. Sections were washed with
TBS buffered saline (pH 7) between each incubation step. Finally, sections were counterstained
with haematoxylin for 20 s and mounted. Slides incubated with non-immune serum from the
species in which the primary antibody was raised instead of the primary antibodies served as
negative controls. Lymphoid tissue of B. constrictor served as a positive control.
For the identification of B cells, RNA-ISH was performed using the RNAscope® technology
(Advanced Cell Diagnostics (ACD), Silicon Valley, USA) and the RNAscope 2.5 Detection Reagent
Kit (Brown) according to the manufacturer’s protocol. All cases were first tested for the suit‐
ability of the material (RNA preservation and quality) with an oligoprobe for mouse ubiquitin
(MUC) (BLAST analysis (NCBI, BLAST) of the MUC oligoprobe sequence to the python bivitattus
genome (accession number XM_015890515.2) identified 87.99% as the highest percentage of
identity). Those yielding good MUC signals were then subjected to RNA-ISH with oligoprobes
coding for B. constrictor CD20 (mRNA Sequence ID XM_007431414.3). Briefly, sections were
heated to 60°C for 1 h and subsequently deparaffinized. Permeabilization was achieved by in‐
cubation in pretreatment solution 1 (RNAscope Hydrogen Peroxide) for 10 min at RT.
Afterwards, the sections were boiled in RNAscope 1× Target Retrieval Reagents solution at
100°C for 15 min, followed by washing in distilled water and ethanol. After digestion with
RNAscope Protease Plus for 30 min at 40°C, sections were hybridized with the oligoprobes at
40°C in a humidity control tray for 2 h (HybEZ Oven, ACD). Thereafter, serial amplification with
different amplifying solutions (AMP1, AMP2, AMP3, AMP4: alternating 15 and 30 min at 40°C)
was performed. Between each incubation step, slides were washed with washing buffer. They
were subsequently incubated with AMP5, AMP6 and DAB at RT for 30 min and 15 min, respec‐
tively. Gill’s haematoxylin served to counterstain the sections that were then dehydrated with
graded alcohol and xylene and coverslipped. Consecutive sections incubated accordingly but
without including the hybridization step served as negative controls.
For TEM, glutaraldehyde buffy coat pellets from 3 adult animals were routinely embedded in
epoxy resin. Toluidine blue-stained semithin sections (1.5 μm) were prepared to select areas of
interest for the preparation of ultrathin sections (75 nm). The latter were contrasted with lead
citrate and uranyl acetate and viewed with a Philips CM10, operating with a Gatan Orius
Sc1000 digital camera (Gatan Microscopical Suite, Digital Micrograph, Pleasanton, USA).
Morphological identification of blood cells
For the identification of the blood cells and to determine their morphological features, previ‐
ous literature was consulted. To identify the blood cells in cytologic smears, we referred to lit‐
erature on other snake families/species, for example, kingsnakes, kobras, blood pythons and
pine snakes (Salakij et al., 2002; Giori et al., 2020; Stacy et al., 2011) and/or other reptile
classes (Moller et al., 2016) as data on the morphological features of blood cells in B. constric‐
tor were not available. The ultrastructural identification of blood cells was also partially based
on findings from previous studies on other reptile species (Salakij et al., 2002; Moller et al.,
2016). However, there was no literature on reptiles that could be referred to for a clear differ‐
entiation and distinction of certain cell types, for example, azurophils and monocytes.
Therefore, we used the ‘monocytic’ features known from mammals, that is, horses and dogs
(Mehta and Sinha, 2018; Pereira et al., 2019) to identify monocytes ultrastructurally. Azurophils
were identified based on their appearance and overall high number in the cytological smear
(Stacy et al., 2011); this was then correlated with their location in the buffy coat and the ex‐
pression of Iba1, a monocyte marker expressed across different orders (Pierezan et al., 2014).
An overall distinction between blood cells of the granulocytic and agranulocytic lineage as ap‐
plied in some former reptile studies (Hü seyin Arıkan, 2014; Jenkins-Perez, 2012) was not
found to be appropriate for the present study. This was due to the fact that monocytes, histori‐
cally belonging to the ‘agranulocytes’ (McMillan and Harris RJ, 2018), exhibited structures in
TEM that were ultrastructurally consistent with intracytoplasmic granules; these have also
been described in humans (Collin et al., 2016).
Haematology
Haematological parameters (total leukocyte count, haematocrit, haemoglobin, mean cell

haemoglobin concentration (MCHC), heterophil, azurophil, lymphocyte, eosinophil, basophil
and monocyte counts) were assessed in 49 individuals (Supplemental Table 1).
Blood smears were prepared and stained with an automated staining instrument (HEMA-TEK
2000 slide stainer, Bayer HealthCare AG, Berlin, Germany), using a modified Wright-Giemsa so‐
lution (Hematek® Stain Pak, Siemens Healthcare Inc., New York, USA) within 6 h of sampling
and air dried. A 100 white blood cell (WBC) differential count was performed by two labora‐
tory technicians with experience in reptilian haematology. Out of the two differential counts,
the mean was calculated to obtain a percentage for each cell type.
The packed cell volume (PCV) was determined by placing blood into a plain glass microhaemat‐
ocrit tube (Arnold Bott AG, Glattbrugg, Switzerland) with one end sealed and spun at 12 000 g
for 5 min using a microhaematocrit centrifuge (Haematocrit 210 Centrifuge, Hettich, Bä ch,
Switzerland).
The haemoglobin concentration was assessed with the cyanmethaemoglobin method and mea‐
sured using a photometer (Photometer LP 400, Dr Lange, Switzerland) after lysis of the red
blood cells and centrifugation of the lysate for 5 min at 12 000 g, followed by colorimetric mea‐
surement at 540 nm. The MCHC was calculated from the PCV and the haemoglobin
concentration.
Total WBC and red blood cell counts were obtained using a 1:200 blood dilution with Natt and
Herrick solution and an improved Neubauer haemocytometer as previously reported (Carisch
et al., 2019).
Clinical chemistry
The following biochemical parameters were measured in the plasma: total protein (TP), choles‐
terol, aspartate transaminase (AST), albumin (ALB), lactate dehydrogenase (LDH), uric acid and
glucose. The tests were performed in 66 (TP, cholesterol, AST, LDH) and 39 (uric acid and glu‐
cose) animals, respectively (Supplemental Table 1).
All parameters were determined by colorimetry with an autoanalyser (Cobas Mira Roche auto‐
analyser, Hoffmann-La Roche Ltd, Basel, Switzerland), using commercially available kits (AST,
cholesterol, LDH, TP, ALB: Diatools AG, Villmergen, Switzerland; uric acid: AxonLab, Dä ttwil,
Switzerland). The glucose concentration was measured using a Roche Cobas c501 analyser
(Roche Diagnostics, Schweiz AG, Rotkreuz, Switzerland).
Corticosterone measurement
In 22 animals (Supplementary Table 1), plasma samples were analysed using a specific enzyme-
immunoassay for corticosterone (Mö stl et al., 2002). A 150-μl aliquot of plasma was extracted
with 3 ml of diethyl ether. After vortexing for 30 min, the plasma ether mixture was frozen at
−20°C. The ether was poured into a new vial and evaporated to dryness, and then the residue
was dissolved in 300-μl assay buffer. The extracted samples were analysed in duplicates. Serial
dilutions of serum samples yielded parallel profiles with the standard curve.
Statistical analysis
Haematological, plasma biochemical and corticosterone values were analysed using Stata 13
(StataCorp. 2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP.). The
level of significance testing was set with a P value of 0.05. Descriptive statistics were applied,
and the data were tested for normality by the Shapiro–Wilk W test. For assessment of age-re‐
lated differences, two age groups were established, young animals (<3 years of age; prior to
definite sexual maturity and reproduction) and adult (≥3 years of age; sexually mature, repro‐
ducing animals).
Normally distributed data were analysed using t-test and ANOVA; non-parametric tests
(Wicoxon rank-sum/Mann–Whitney) were used for data that were not normally distributed.
Geometric and arithmetic mean, including confidence intervals, were determined for logarith‐
mically transformed and raw data, respectively. Medians were reported where non-parametric
tests are being used. Linear regression was used and linearity was confirmed using the qnorm
plot and the Shapiro–Wilk W test on residuals.
Variables where data were normally distributed include the percentage of lymphocytes and
azurophils, haematocrit, haemoglobin, MCHC, total protein, albumin, and glucose. Logarithmic
transformation was applied on data for total number of leukocytes, number of lymphocytes,
eosinophils, percentage of heterophils, eosinophils, monocytes and basophils, LDH, AST, urea,
cholesterol and corticosterone. Nonparametric tests were used for number of azurophils, het‐
erophils, monocytes and basophils and uric acid.
Results
Animals and disease/infection state
Thirty-one animals were young (<3 years of age; juvenile and subadult; Myers et al., 2022); the
remaining 48 animals were adult (≥3 years of age) (Table 1). Overall, the age of the animals
ranged from 3 months to 20 years, with an average of 4.52 years. Both age groups comprised
male and female animals in almost equal proportions; however, for 14 snakes, information on
the sex either was not provided or could not be determined due to their young age
(Supplementary Table 1).
Table 1
Leukocyte numbers in the peripheral blood (per μl) of clinically healthy captive B. constrictor.
Cell type Female Male Test Total
<3 ≥3 Total <3 ≥3 Total para
years years years years meters
(n) (n) (n) (n) (n) (n) (n)

Mean* Mean Mean Mean Mean Mean Mean
(95% (95% (95% (95% (95% (95% (95%
CI) CI) CI) CI) CI) CI) CI)
Leukocytes (3) (16) (20) (6) (11) (17) t= (49)

(2)
12.004 9.561 9.101 7.418 7.793 7.659 −0.6769, 8.306
× 103/μl (7.009– (5.851– (5.945– (4.125– (5.210– (5.719– df = 35, (6.768–
20.558) 15.624) 13.932) 13.338) 11.657) 10.257) P= 10.193)
0.5029
t = 0.4162, df = 17, P t = −0.1660, df = 15,

= 0.685 P = 0.8704
Lymphocytes (3) (16) (20) (6) (11) (17) t= (49)

(2)
8.822 3.877 4.020 3.640 2.678 2.984 −0.9083, 3.480
× 103/μl (3.142– (2.037– (2.295– (1.831– (1.680– (2.107– df = 35, (2.679–
24.765) 7.381) 7.043) 7.238) 4.270) 4.227) P= 4.522)
0.3699
t = 1.1424, df = 17, P t = 0.8873, df = 15, P

= 0.2691 = 0.3889
Azurophils (3) (3) (16) (20) (6) (11) (17) z = 0.411, (49)
3
× 10 /μl 1.300 2.245 1.935 1.275 2.590 2.340 P= 1.880
(0.150– (1.222– (0.637– (0.185– (1.344– (1.084– 0.6807 (1.475–
**
2.240) 4.996) 3.782) 5.756) 4.140) 3.319) 2.499)
z = −1.230, P = z = −1.407, P =
0.2188 0.1594
Heterophils (3) (16) (20) (6) (11) (17) z= (49)

(3)
1.250 2.090 1.900 1.390 2.290 1.740 −0.305, 1.610
3
× 10 /μl (0.690– (0.963– (0.899– (0.290– (0.806– (0.830– P= (1.040–
**
1.460 3.255) 2.776) 2-280) 3.777) 2.399) 0.7605 2.132)
z = −1.342, P = z = −0.905, P =
0.1797 0.3654
Eosinophils (3) (10) (14) (3) (7) (10) t= (34)

(2) 0.161 0.144 0.147 0.129 0.062 0.077 −1.3731, 0.151
3
× 10 /μl (0.013– (0.049– (0.069– (0.009– (0.032– (0.042– df = 22, (0.098–
2 010) 0 421) 0 314) 1 804) 0 117) 0 140) P= 0 231)
*The measures of position depend on data distribution: Arithmetic mean (1) for normally distributed data,
geometric mean (2) for logarithmic transformation and median (3) for non-normal distribution.
**Lower (upper) confidence limit held at minimum (maximum) of sample.
A) Leukocyte concentrations in female and male B. constrictor, further divided according to the age groups
(young: < 3 year; adult: ≥ 3 years). B) Leukocyte concentrations in young (< 3 years) and adult (≥ 3 years) B.
constrictor, further divided by sex.
Table 2
Relative leukocyte percentages of clinically healthy captive B. constrictor.
Cell type Female Male Total
<3 ≥3 Total <3 ≥3 Total

years years years years
(n) (n) (n) (n) (n) (n) Test (n)

Mean* Mean Mean Mean Mean Mean para Mean
(95% CI) (95% (95% (95% (95% (95% meters (95%
CI) CI) CI) CI) CI) CI)
Lymphocytes (3) (16) (20) (6) (11) (17) t= (49)

(1)
(%) 74.5% 45.13% 49.20% 51.42% 35.73% 41.26% −1.3089, 45.17%
(35.70– (34.95– (39.44– (33.88– (28.34– (33.59– df = 35, (40.26–
100.00) 55.30) 58.96) 68.95) 43.12) 48.94) P= 50.09)
0.1991
t = 2.4938, df = 17, P t = 2.3456, df = 15, P

< 0.05 < 0.05
(1)
Azurophils (3) (16) (20) (6) (11) (17) t= (49)
(%) 11.50% 26.38% 22.98% 24.83% 31.27% 29.00% 1.3813, 25.95%
(13.99– (18.61– (15.87– (9.46– (26.48– (23.64– df = 35, (22.21–
36.99) 34.14) 30.08) 40.20) 36.06) 34.36) P= 29.69)
0.1759
t = −1.6721, df = 17, t = −1.2361, df = 15,

P = 0.1128 P = 0.2354
Heterophils (3) (16) (20) (6) (11) (17) t= (49)

(2)
(%) 9.00% 18.32% 17.02% 14.28% 23.13% 19.51% 0.6696, 17.76%
(2.011– (13.17– (12.49– (7.11– (17.14– (14.60– df = 35, (15.08–
40.27) 25.47) 23.19) 28.68) 31.22) 26.09) P= 20.91)
0.5075
t = −1.8303, df = 17, t = −1.7953, df = 15,

P = 0.0848 P = 0.0928
Eosinophils (3) (10) (14) (3) (7) (10) t= (34)

(2)
1.36% 1.46% 1.63% 1.82% 0.87% 1.08% −1.2964, 1.83%
(%) (0.16– (0.88– (1.01– (0.46– (0.51– (0.69– df = 22, (1.33–
11.84) 2.43) 2.61) 7.22) 1.47) 1.72) P= 2.51)
0.2083
t = −0.1540, df = 11, t = 1.8867, df = 8, P =

P = 0.8804 0.0959
Monocytes (2) (2) (15) (18) (6) (11) (17) t= (47)

(%) 1 73% 2 36% 2 22% 3 10% 2 76% 2 87% 1 2452 2 50%
*The measures of position depend on data distribution: Arithmetic mean (1) for Normally distributed,
Geometric mean (2) for logarithmic transformation and median (3) for non-normal distribution
A) Relative leukocyte percentages in female and male B. constrictor, further divided according to the age
groups (young: < 3 year; adult: ≥ 3 years). B) Relative leukocyte percentages in young (< 3 years) and adult (≥
3 years) B. constrictor, further divided by sex.
Haematology
Haematological data were collected from 49 animals, 22 young and 27 adult boas. The differen‐
tial WBC counts for the two age groups and for male and female snakes are provided in
Tables 1–3.
Table 3
Blood parameters (haematocrit, haemoglobin, MCHC) of clinically healthy captive B. constrictor, divided by
sex (upper table) and age (lower table)
Female Male
Para meter <3 years ≥3 Total <3 years ≥3 Total Test Total
years years para
meters
*
(n)Mean (n) (n) (n) (n) (n) (n)
(95% CI) Mean Mean Mean Mean Mean Mean
(95% (95% (95% (95% (95% (95%
CI) CI) CI) CI) CI) CI)
Haematocrit (3) (14) (18) (6) (8) (14) t= (44)

(1)
24.333 27.429 26.278 28.833 30.500 29.786 2.4211, 26.023
(10.208– (25.864– (24.204– (26.313– (26.478– (27.519– df = 30, (24.470–
38.459) 28.993) 28.351) 31.353) 34.522) 32.052) P < 0.05 27.575)
t = −1.4892, df = 15, P t = −0.7739, df = 12,

= 0.1572 P = 0.4540
Haemoglobin (3) 7.033 (16) (20) (6) (11) (17) t= (49)

(1)
(3.174– 8.101 7.805 8.417 9.473 9.100 2.8486, 8.012
10.892) (7.518– (7.192– (7.848– (8.347– (8.352– df = 35, (7.546–
8.694) 8.418) 8.985) 10.598) 9.848) P < 0.01 8.479)
t = −1.4631, df = 17, P t = −1.4830, df = 17,

= 0.1617 P = 0.1588
MCHC (1) (3) I16) (20) (6) (11) (17) t= (49)

29.333 29.250 29.450 29.333 31.091 30.471 1.0088, 30.429
(27.899– (27.593– (28.096– (27.166– (28.605– (28.794– df = 35, (29.567–
30.768) 30.907) 30.804) 31.501) 33.577) 32.148) P= 31.290)
0.3200
t = 0.0452, df = 17, P t = −1.0662, df = 15,

= 0.9644 P = 0.3032
<3 years ≥3 years
Female Male Total Female Male Total
(n) Mean* (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Haematocrit (3) (6) (21) (14) (8) (22) t= (44)

(1)
24.333 28.833 23.857 27.429 30.500 28.545 −3.5187 26.023
(10.208– (26.313– (21.635– (25.864– (26.478– (26.854– df = 41, (24.470–
38.459) 31.353) 26.079) 28.993) 34.522) 30.236) P < 0.01 27.575)
t = 1.7413, df = 7, P = t = 1.9316, df = 20, P
*
The measures of position depend on data distribution: Arithmetic mean (1) for Normally distributed,
Geometric mean (2) for logarithmic transformation and median (3) for non-normal distribution
Lymphocytes were the most abundant WBC in all animals, regardless of age and sex, followed
by azurophils and heterophils; in contrast, the percentage of eosinophils, monocytes and ba‐
sophils was always low and never exceeded 3% (Fig. 1 A, B). The younger animals (<3 years)
showed a significantly lower percentage of eosinophils (geometric mean = 2.64%, CI = 1.63–
4.28) than the adult animals (geometric mean = 1.18%, CI = 0.83–1.68, t = 2.895, df = 31, P <
0.01) (Fig. 1A).
Figure 1
Age- and sex-associated differences in blood parameters of healthy B. constrictor. A, B. Distribution of

leukocyte subpopulations. A. Lymphocytes, azurophils and heterophils are the leukocyte subpopulations
with the highest concentration in young (<3 years) and adult (≥3 years) B. constrictor. The concentration of
eosinophils is significantly higher in the young animals. Box and whisker plots, *P < 0.05. B. Lymphocytes,
azurophils and heterophils are the dominant leukocyte subpopulations in both female and male B. constrictor.
Data provided as log-transformed values. C. Age associated differences. The percentage of lymphocytes
shows a linear association and decreases with age. D. Sex associated differences. Male snakes show higher
cholesterol levels than female snakes in both age groups. Box and whisker plots, *P < 0.05.
The percentage of lymphocytes showed a linear association with age and sex: R2 = 0.2421,
F(2.31) = 4.95, P < 0.05 (lymphocyte percentage for female animals = 43.38 + 9.62 + (−2.006 ×
age), and for male animals = 43.38 + (−2.006 × age)) (Fig. 1C). The percentage of heterophils
was significantly associated with the age group: R2 = 0.1809, F(2,33) = 3.64, P < 0.05, and higher
in the adult boas.
Adult animals had a significantly higher haematocrit (mean = 28.56, CI = 26.85–30.24) and
haemoglobin (mean = 8.66, CI = 8.01–9.26) than the young animals (mean = 23.86, CI = 21.64–
26.08 with t = −3.5, df = 41, P < 0.01 for haematocrit levels, and mean = 7.31, CI = 6.7–7.94 with
t = 3.21, df = 46, P < 0.01 for haemoglobin levels). Both parameters were significantly higher in
males (mean = 29.79, CI = 27.52–30.05 for haematocrit, and mean = 9.1, CI = 8.35–9.85 for
haemoglobin levels) than in females (mean = 26.28, CI = 24.2–28.35 with t = 2.42, df = 30, P <
0.05 for haematocrit, and mean = 7.81, CI = 7.18–8.42 with t = 2.85, df = 35, P < 0.01 for
haemoglobin levels). Haemoglobin levels also showed a linear association with sex: R2 =
0.2064, F2,31 = 40.03, P < 0.05 (female: haemoglobin = 9.647–1.078 + 0.089 × age; male:
haemoglobin = 9.647 + 0.089 × age).
All other haematological parameters did not vary significantly with age and sex (Tables 1–3).
Identification and morphological features of blood cells
The light microscopic examination of the buffy coats revealed no distinct layering of the leuko‐
cytes (Fig. 2), confirming a previous report (Fontes Pinto et al., 2018). Apart from heterophils,
which could be identified due to their prominent intracytoplasmic granules (Fig. 2B), differenti‐
ation of the leukocyte subpopulations based on their morphology was not possible, as cell bor‐
ders, nuclear and/or cytoplasmic features could not be fully discerned (Fig. 2A). However, the
immunohistochemical staining and RNA-ISH for T and B cells and monocytes (Fig. 2B-D), the
cytological examination of the blood smears and the ultrastructural examinations of the buffy
coat pellets allowed further identification of the different blood cell populations (Figs. 3-5).

Figure 2
Buffy coat of peripheral blood of healthy B. constrictors after formalin fixation and paraffin embed‐
ding. A. HE-stained section of a buffy coat. Top: The blood cells do not arrange in distinct layers. Erythrocytes
accumulate as a large aggregate below the buffy coat (asterisk). Bar = 100 μm. Bottom: The higher magnifica‐
tion shows the presence of small groups of erythrocytes (arrowhead) and small fibrinous aggregates (arrows)
between the leukocytes. Bar = 25 μm. B. Iba-1, a confirmed marker of monocytes across animal classes, is ex‐
pressed by numerous cells primarily at the bottom of the buffy coat (top, arrow). Due to their abundance, the
Iba1-positive cells are interpreted as both monocytes and azurophils. Bottom: Strongly Iba1-positive
monocytes/azurophils interspersed with numerous Iba1-negative heterophils, identified based on the pres‐
ence of their distinct cytoplasmic granules (inset, arrow). Immunohistochemistry, haematoxylin counter‐
stain. Bars = 100 μm (top) and 10 μm (bottom and inset). C. CD3, a confirmed marker of T cells across animal
classes, is expressed by numerous cells primarily at the top of the buffy coat (top, arrow). Bottom: A higher
magnification confirms the presence of abundant positive cells, interspersed with some aggregates of throm‐
bocytes (arrow). Immunohistochemistry, haematoxylin counterstain. Bars = 100 μm (top) and 10 μm (bot‐
tom). D. RNA-ISH for CD20, a confirmed marker of B cells across animal classes, shows a signal in a low
number of cells throughout the buffy coat, either as individual (arrowheads) or small groups (arrow) of round
cells. RNA-ISH, haematoxylin counterstain. Bar = 10 μm.
Figure 3
Morphological features of erythrocytes and thrombocytes in the peripheral blood of healthy B. con‐
strictors. A, B. Erythrocytes. A. Cytological specimen. Erythrocytes are large, oval cells with a central oval
nucleus and homogenous eosinophilic cytoplasm. B. TEM. Erythrocytes are elongated cells with uniformly
electron-dense cytoplasm. The nucleus is round, with clumped heterochromatin that is arranged peripherally,
along the nuclear membrane. C, D. Thrombocytes. C. Cytological specimen. Aggregate of four round thrombo‐
cytes, with small cytoplasmic pseudopodia and a round central nucleus (‘round’ form). There is also one
thrombocyte with an ‘elongated’ form (arrowhead), a clear juxtanuclear cytoplasmic halo (arrow) and an oval
nucleus. D. TEM of three thrombocytes. The cells are dominated by a central, irregularly outlined nucleus, a
small amount of cytoplasm that contains small electron-dense vacuoles (arrowheads) and elongated to round
more electron-lucent structures (‘canalicular structures’, arrow). A, C: May–Grü nwald–Giemsa stain, bars =
10 μm; B, D: TEM, bars = 5 μm (C) and 2 μm (D).
Figure 5
Morphological features of azurophils and monocytes in the peripheral blood of healthy B. constrictor.
A, B. Azurophils. A. Cytological specimen showing an azurophil, a round cell with abundant variably ba‐
sophilic to intensely eosinophilic cytoplasm that contains a few small clear vacuoles (arrow). The central nu‐
cleus is round, with clumped chromatin. B. Ultrastructure of an azurophil. The cell exhibits small cytoplas‐
mic projections (small arrow) and variably sized cytoplasmic vacuoles that contain finely granular material
of low electron density (arrow). C, D. Monocytes. C. Cytological specimen showing a monocyte. The round
cell has abundant pale basophilic and moderately granular cytoplasm with a variable number of small vac‐
uoles (arrow) and small cytoplasmic protrusions. The nucleus is eccentric and slightly indented. D. TEM im‐
age of a monocyte. There are several fine cytoplasmic protrusions (small arrows); the cytoplasm contains
abundant organelles, amongst them small electron-dense round to elongated granules (asterisk), mitochon‐
dria (arrowhead), rough endoplasmic reticulum and lysosomes (arrows). The nucleus is eccentric and in‐
dented, with marginalized clumped chromatin and a distinct nucleolus. E, F. Direct comparison of azurophils
and monocytes. E. In the cytological specimens, azurophils (A) present a darker, more homogenous, largely
bright eosinophilic cytoplasm and a central, round nucleus. In contrast, the monocyte (M) has pale basophilic
cytoplasm and an eccentric, indented nucleus. F. Ultrastructurally, the cytoplasm of the azurophil (A) is rich
in vacuoles with variable content (asterisk), whereas the cytoplasm of the monocyte (M) is rich in organelles
(mitochondria, rER, lysosomes) and contains abundant small electron dense granules. G, H. Lymphocytes.G.
Cytological specimen with a lymphocyte, presenting as a small round cell with a round nucleus containing
highly clumped chromatin; the cytoplasm is scant and basophilic. H. The cells exhibit scant cytoplasm with
few mitochondria (arrow) and a round nucleus with large clumps of mainly peripheral heterochromatin. A, C,
E, G: May–Grü nwald–Giemsa stain, bars = 5 μm; B, D, F, H: TEM, bars = 2 μm.
Erythrocytes The red blood cells formed a large aggregate below the buffy coat (Fig. 2A).
Together with occasional variably sized fibrin clots, they were also present as small groups
throughout all layers (Fig. 2A). Cytologically, erythrocytes presented as large oval uniform cells
of consistent size (length: 12–16 μm, width: 5–7 μm), with a centrally located oval nucleus (
Fig. 3A). Ultrastructurally, they were characterized by their elongate shape and uniformly elec‐
tron-dense cytoplasm. Nuclei were round and uniform, with clumped heterochromatin that was
arranged peripherally, along the nuclear membrane (Fig. 3B).
Thrombocytes Thrombocytes were found on the top of the buffy coat. In the cytological speci‐
mens, they were 8–10 μm in diameter and varied in shape. Most were round with small cyto‐
plasmic pseudopodia and a round central nucleus and often found in aggregates; others were
elongate and exhibited a clear juxtanuclear cytoplasmic halo and an oval nucleus with dense
chromatin (Fig. 3C). At ultrastructural level, thrombocytes were characterized by small elec‐
tron-dense vacuoles and elongated to round electron loose structures (canalicular structures)
in the cytoplasm (Israels SJ, 2007) and a round central nucleus (Fig. 3D).
Heterophils represented the most abundant granulocytes (Fig. 1A). They could be readily
identified in the cytological specimens, as round cells with an eccentric round nucleus and a
high number of sometimes refringent cytoplasmic granules with variable staining properties (
Fig. 4A). Their size ranged between 15 and 25 μm in diameter. In the buffy coats, heterophils
were mainly found in the bottom layer (Fig. 2B). TEM showed that the cytoplasm of heterophils
is packed with granules; these are heterogeneous in shape, about 0.5–1 μm in diameter and of
moderate to high electron density. Some heterophils also exhibited electron lucent vacuoles
that contained irregularly shaped material (phagosomes/phagolysosomes) (data not shown). A
few distinct organelles such as mitochondria were also observed. The nucleus was round, with
marginalized clumps of heterochromatin (Fig. 4B).
Figure 4
Morphological features of heterophils, basophils and eosinophils in the peripheral blood of healthy
B. constrictor. A, B. Heterophils. A. Cytological specimen, showing a heterophil. The cytoplasm of the round
cell is brightly eosinophilic and contains a moderate number of variably distinct granules (arrow); the nu‐
cleus is oval and eccentric. B. TEM shows abundant round electron dense cytoplasmic granules of variable
size (arrows). The round nucleus is slightly peripherally located and shows marginalized clumps of hete‐
rochromatin. C, D. Basophils.C. Cytological specimen showing a basophil. The cell is slightly ovoid and ex‐
hibits abundant cytoplasm that is entirely filled by numerous round, distinct, intensely basophilic granules.
D. Ultrastructurally, the granules are round and contain homogenous, highly electron-dense material (arrows).
A few mitochondria (arrowheads) are also obvious. The nucleus is located peripherally and contains clumped
heterochromatin. E, F. Eosinophils.E. Cytological specimen with an eosinophil. This round cell is smaller
than heterophils and basophils, the cytoplasm is entirely filled by small indistinct darkly basophilic and
eosinophilic granules. The nucleus is located in the periphery and appears slightly indented. F. TEM shows
numerous round to elongated, variably sized granules in the cytoplasm filled with variably electron-dense
material (arrows). Some granules show a central core (C) and a matrix (M). The nucleus has an irregular out‐
line and abundant clumped heterochromatin. Arrowhead: mitochondrium. A, C, E: May–Grü nwald–Giemsa
stain, bars = 5 μm; B, D, F: TEM, bars = 2 μm.
Basophils were also readily identified in the cytological specimens, due to their abundant cyto‐
plasm filled with numerous round, variably sized, intensely basophilic granules that occasion‐
ally masked the nucleus (Fig. 4C). They were 20–25 μm in diameter, round to oval, with a cen‐
tral to eccentric round nucleus. Ultrastructurally, the granules were 0.2–1 μm in diameter,
round and of high electron density (Fig. 4D). Like in heterophils, the cytoplasm also contained
a few distinct organelles such as mitochondria, and the nucleus was round, with marginalized
clumps of heterochromatin.
Eosinophils were the smallest granulocytes (5–8 μm in diameter) in the cytological specimens
(Fig. 4E). They were also round. The cytoplasm was more dense than in heterophils and con‐
tained abundant, variably distinct dark basophilic and eosinophilic granules. The nucleus was
usually hyperchromatic and located in the periphery. TEM revealed an eccentric, ovoid nucleus
with a moderate amount of peripheral heterochromatin. The abundant round to elongate cyto‐
plasmic granules had a variably electron-dense homogenous content. Again, a few distinct or‐
ganelles such as mitochondria were also identified in the cytoplasm (Fig. 4F).
Azurophils and monocytes While monocytes in reptiles are generally thought to be morpholog‐
ically and functionally similar to their mammalian counterpart, azurophils appear to be unique
to reptile species (Vickie Joseph, 2015; Stacy et al., 2011). Depending on the reptile order, how‐
ever, they are described as morphologically (and possibly functionally) similar to both granulo‐
cytes and monocytes (Stacy et al., 2011; Vickie Joseph, 2015). As previously described (Stacy et
al., 2011), azurophils and monocytes could be readily discerned in the cytological specimens (
Fig. 5). Azurophils were round, 15–20 μm in diameter, with often brightly eosinophilic cyto‐
plasm that contained occasional clear vacuoles. The nucleus was generally round and, different
from basophils and eosinophils, centrally located, with clumped chromatin (Fig. 5A).
Monocytes presented as roundish cells, with a slightly broader size range (12–25 μm in diame‐
ter). They exhibited small pseudopodia and a pale basophilic and moderately granular cyto‐
plasm with or without vacuoles. The nucleus was central or eccentric and sometimes indented (
Fig. 5C). Azurophils were by far more abundant than monocytes in the cytological specimens
and could readily be differentiated from the latter due to the more eosinophilic cytoplasm (
Fig. 5E).
In HE-stained buffy coat sections, however, monocytes and azurophils could not readily be dis‐
cerned (Fig. 2A). Staining for Iba1, a marker of monocytes in a wide range of animal
classes/species (Pierezan et al., 2014) showed positive cells primarily in the bottom layer, sug‐
gesting these to be monocytes (Fig. 2B). However, considering the morphology of the Iba1-pos‐
itive cells and their number and proportion in the haematological assessment, it appears likely
that these represented both monocytes and azurophils (Fig. 2B). In contrast, heterophils, which
could be readily discerned based on their distinct granules, were clearly Iba1 negative (Fig. 2B
bottom, inset).
Also at ultrastructural level, monocytes and azurophils could readily be discerned (Fig. 5). The
more abundant cells that were neither granulocytes nor lymphocytes, hence interpreted as
azurophils, were 8–10 μm in diameter, with small cytoplasmic projections and vacuoles of vari‐
able number and size (0.5–2 μm) that contained finely granular material of low electron den‐
sity (Fig. 5B and F). They occasionally contained cytoplasmic vacuoles filled with irregularly
shaped material (phagosomes/phagolysosomes) and had a round nucleus with a significant
amount of clumped heterochromatin. The second, less frequently encountered cells, hence in‐
terpreted as monocytes, were 8–12 μm in diameter and had an eccentric, often indented nu‐
cleus with a lesser amount of heterochromatin. Their cytoplasm formed thin projections and
contained a high number of organelles, that is, mitochondria, rough endoplasmic reticulum and
lysosomes, as well as abundant small electron-dense granules, and (Fig. 5D and F).
Lymphocytes In the cytological specimens, lymphocytes presented as small- to medium-sized

cells (5–10 μm in diameter) with a roundish outline and a central, round, purple to basophilic
nucleus containing clumped chromatin (Fig. 5G). Immunohistochemical staining for CD 3 identi‐
fied abundant T cells primarily at the top of the buffy coat (Fig. 2C). In contrast, B cells, identi‐
fied based on the transcription of the B cell marker CD20 as shown by RNA-ISH, were rare and
found individually or in small groups in the mid and upper layers of the buffy coat (Fig. 2D).
This suggests that T cells are far more abundant than B cells in the peripheral blood of B. con‐
strictor. The ultrastructural examination confirmed that lymphocytes have scant cytoplasm and
few organelles, such as mitochondria, and a round nucleus with large clumps of mainly periph‐
eral chromatin (Fig. 5F).
Biochemical parameters and corticosteroid levels
Biochemical parameters (total protein, albumin, cholesterol, uric acid, glucose, urea, AST and
LDH) were assessed in 66 animals, 27 young and 39 adult boas. The biochemical data for the
two age groups and for male and female snakes (26 males and 25 females) are provided in
Table 4.
Table 4
Biochemical parameters and corticosterone levels of clinically healthy captive B. constrictor.
Female Male
Para-meter <3 ≥3 Total <3 ≥3 Total Test Total

years years years years para
meters
(n) (n) (n) (n) (n) (n) (n)

*
Mean Mean Mean Mean Mean Mean Mean
(95% (95% (95% (95% (95% (95% (95%
CI) CI) CI) CI) CI) CI) CI)
Total protein (5) (20) (25) (7) (19) (26) t= (66)

(1) 38.680 53.030 50.160 44.843 53.468 51.146 0.2809, 48.533
(20.048– (47.864– (44.805– (34.782– (47.713– (46.261– df = 49, (45.577–
57.312) 58.196) 55.515) 54.904) 59.224) 56.032) P= 51.489)
0.7800
t = −2.4273, df = 23, t = −1.6696, df = 24,

P < 0.05 P = 0.1080
Albumin (1) (5) (20) (25) (7) (19) (26) t= (66)

21.860 29.900 28.292 25.971 30.416 29.219 0.5115, 27.453
(12.622– (26.936– (25.323– (19.591– (28.077– (26.919– df = 49, (25.815–
31.098) 32.864) 31.261) 32.352) 32.755) 31.519) P= 29.091)
0.6113
t = −2.4591, df = 23, t = −1.8487, df = 24,

P < 0.05 P = 0.0769
LDH (2) (5) (20) (25) (7) (19) (26) t= (66)

68.367 112.309 101.696 115.381 100.631 104.406 0.1215, 105.845
(37.131– (75.385– (72.801– (58.512– (70.036– (77.585– df = 49, (88.532–
125.880) 167.319) 142.059) 227.522) 144.530) 140.497) P= 126.543)
0.9038
t = −1.2396, df = 23, t = 0.4138, df = 24, P

P = 0.2276 = 0.6827
AST (2) (5) (20) (25) (7) (19) (26) t= (66)

18.969 16.619 17.065 19.770 19.258 19.394 0.6081, 18.907
(4.994– (11.041– (11.797– (12.644– (14.241– (15.333– df = 49, (15.821–
72.056) 25.016) 24.685) 30.912) 26.042) 24.532) P= 22.597)
0.5459
t = 0.2900, df = 23, P t = 0.0999, df = 24, P

= 0.7744 = 0.9212
Cholesterol (2) (5) (20) (25) (7) (19) (26) t= (66)

*The measures of position depend on data distribution: Arithmetic mean (1) for Normally distributed,
Geometric mean (2) for logarithmic transformation and median (3) for non normal distribution
** Lower (upper) confidence limit held at minimum (maximum) of sample
(A) Biochemical parameters and corticosterone levels for young (< 3 years) and adult (≥ 3 years) B. constric‐
tor, further divided by sex. (B) Biochemical parameters and corticosterone levels for young (< 3 years) and
adult (≥ 3 years) B.constrictor, further divided by sex.
Adult animals had significantly higher total protein (mean = 53.41, CI = 49.82–57) and albumin
(mean = 30.37, CI = 28.55–31.19) levels than the young snakes (mean = 41.03, CI = 37.37–44.7
with t = −4.7, df = 64, P < 0.0001 for total protein, and mean = 22.97, CI = 20.74–25.19 with t =
−5.22, df = 64, P < 0.0001 for albumin). The total protein level also showed an association with
age (R2 = 0.1483, F2,48 = 4.18, P < 0.05), as older snakes showed higher levels than young
snakes.
Male snakes generally showed higher cholesterol levels than female snakes regardless of age
(young animals: males geometric mean = 2.82, CI = 1.96–4.07, females geometric mean = 1.56,
CI = 0.858–2.85, with t = 2.33, df = 10, P < 0.05; adult animals: males geometric mean = 3.12, CI =
2.21–4.14, females geometric mean = 1.94, CI = 1.4–2.66, with t = −2.13, df = 37, P < 0.05); this is
illustrated in Figure 1D. The association of cholesterol levels with sex was also confirmed (R2 =
0.1416, F2,48 = 3.96, P < 0.05).
All other biochemical parameters as well as the corticosterone levels did not vary significantly
with age and sex (Table 4).
Discussion
This study aimed to establish reference data for the peripheral blood of captive B. constrictor. A
wide range of biochemical and haematologic variables, plasma corticosterone levels as well as
the morphology of the peripheral blood cells were investigated. An age and sex group distinc‐
tion was applied due to the commonly known fact that regardless of animal classes or species,
total WBC concentration can differ significantly between young and adult animals and between
sexes, the former possibly being related to sexual maturity (Wood, 2014; Raskin et al., 2004).
Multiple studies in mammals, birds and fewer in reptiles have highlighted the importance of
age-related reference values for the evaluation of leukograms (Lee et al., 2020; Fonseca
Sarmiento et al., 2018; Trimboli et al., 2020; Dolka et al., 2014). We chose 3 years as the cut-off,
because captive B. constrictor are known to reach sexual maturity around 3 years of age. This
does not necessarily apply to their wild counterparts, as here, the onset of reproductive activity
is strongly dependent on the season (Bertona and Chiaraviglio, 2003).
In the wild, B. constrictor can be found in the tropical forests of Central and South America on
altitudes between 0 and 1500 metres above sea level, where there are strong seasonal changes
in precipitation intensity (dry season: March to November; wet season: December to February)
and an ambient temperature that does not fluctuate substantially. Hence, previous studies on
the haematological and biochemical parameters of wild reptiles comprized two sampling time
points (winter/summer season) (el Masri et al., 1995; Holding et al., 2014; Hussein et al., 1979;
Leceta et al., 1989; Leceta and Zapata, 1985; Silva et al., 2011; Machado et al., 2006). Such an
approach was not taken in our study, which included samples collected at ad hoc time points
during the year, as to our knowledge the housing conditions of captive snakes in Switzerland
and Germany do not mimic seasonal variations. While maintenance guidelines for B. constrictor
are closely adapted to the conditions in the wild and often recommend 50–80% humidity and
an air temperature of 24–32°C (Hedley, 2022), personal communication with breeders indi‐
cates that B. constrictor is commonly held at the same temperature throughout the year, with
slight individual temperature differences between day and night.
The haematological examination in the present study yielded leukocyte subset concentrations
that overall aligned with those reported for captive and wild boid snakes so far (Machado et al.,
2006; Fonseca Sarmiento et al., 2018; Jenkins-Perez, 2012). In comparison to previous reports
on captive and wild boas, our total blood leukocyte numbers were relatively high (in the upper
third of the values published so far). However, the WBC numbers were similar to those shown
in the few other studies on captive B. constrictor in which this ‘leukocytosis’ was speculated to
be related to the stress induced by captivity (Machado et al., 2006; Brenner et al., 2002).
Interestingly, a study on wild boids (Corallus hortulanus) indeed reported notably lower WBC
counts (in the lower third of the values published so far) (Quadrini et al., 2018).
In the most common domestic mammal species, neutrophils (dogs, cats, horses etc.) or lympho‐
cytes (pigs, cows) are the dominating leukocyte subpopulations in the peripheral blood of
healthy individuals, with monocytes, eosinophils and basophils comprising a noticeably smaller
proportion (Wood, 2014). In reptiles, lymphocytes (comprising up to 80% of all leukocytes),
heterophils and azurophils, the latter representing a cell type unique to reptiles (Stacy et al.,
2011), have been described as the most frequent leukocyte subpopulations. This was con‐
firmed by the present study that identified lymphocytes as the most abundant leukocyte sub‐
type regardless of age and sex. We also observed a correlation of the lymphocyte proportion
with age, as younger snakes presented a higher percentage of circulating lymphocytes. This is
not surprising considering that young mammals (ruminants, dogs etc.) normally have higher
lymphocyte concentrations than older animals (Wood, 2014). As it does not represent a patho‐
logic process, this physiologic variation is often termed ‘pseudolymphocytosis’ of young animals
(Boes et al., 2017). In humans, the establishment of the adaptive immune system is initiated af‐
ter birth, and the juvenile haematopoiesis becomes lymphoid biassed, which correlates with an
increase in the total number of T lymphocytes by thymic expansion (Holcar et al., 2015). The
results of the present study, as well as those of previous studies on other reptile species
(Fonseca Sarmiento MVZ et al., 2018; Page-Karjian et al., 2021; Casal et al., 2009), indicate a
similar phenomenon also in the class reptilia. Interestingly, the adult boas of the present study
showed a higher lymphocyte percentage than a cohort of captive amazon tree boas previously
studies (Corallus hortulanus) (Quadrini et al., 2018). Whether this reflects real differences be‐
tween closely related species remains unclear but would be suggested, because all examined
animals were clinically healthy, without evidence of inflammatory processes, infectious disease
or ecdysis, which rules out most of the reported causes of lymphocytosis in reptiles (Stacy et
al., 2011; Vickie Joseph, 2015).
In contrast to the lymphocyte percentages, we observed an increase in the percentage of het‐

erophils with age. This finding is in accordance with a study performed on Brazilian wild B. con‐
strictor (Fonseca Sarmiento et al., 2018). In humans, haematopoiesis becomes myeloid biassed
with increasing age and sexual maturation, an effect that is regulated by the influence of extrin‐
sic and intrinsic factors on the haematopoietic stem cells and multipotent progenitor cells
(Wang et al., 2022; Ho et al., 2019; Heo et al., 2015).
We observed significantly higher haematocrit and haemoglobin levels in adult and in male
snakes. In mammals, the haematocrit is also known to increase with age/maturity (Cornell et
al., 2017; Eklom and Lill, 2006), a fact that has been attributed to developmental changes in the
haematopoietic system (Sealander, 1965). Studies on humans and other mammals have also re‐
ported higher haemoglobin levels in male individuals; this is thought to be due to the stimula‐
tory effect of androgens on the bone marrow (Wahed and Dasgupta, 2015). Interestingly,
lower haematocrit and haemoglobin values were reported in adult compared to juvenile wild
B. constrictor from Colombia (Fonseca Sarmiento et al., 2018); yet, unfortunately, information
on exact age and age group definition was not provided for these animals. There is an overall
paucity of data on the age-related changes of haematological variables in reptiles in general
and a direct comparison with mammals might be inappropriate, due to differences in evolu‐
tionary development. As an example, the overall life span of reptile erythrocytes has been
stated to be 600–800 days, depending on the species (Vickie Joseph, 2015), whereas it is
around 20–160 days in mammals (Vá cha and Znojil, 1981). Therefore, more studies, with a va‐
riety of reptile species and larger any cohorts of different age groups are needed to consoli‐
date the findings of this and previous studies.
Overall, the biochemical values obtained from the present boa constrictor cohort were in line
with those formerly reported for this species (Chiodini and Sundberg, 1982). Also, the signifi‐
cantly higher total protein levels in the older snakes are consistent with the relative total pro‐
tein increase with age and weight that has been reported in turtles, tortoises and iguanas
(Casal et al., 2009; Harr et al., 2001; Dickinson et al., 2002). We observed variable cholesterol
levels observed in our group of snakes. Similar to observations in Nile crocodiles (Crocodylus
niloticus) and loggerhead sea turtles (Caretta caretta), we found the these to be higher with age
and in males (Rousselet et al., 2013; Morpurgo and Gelman, 1991). In humans, decreased low
density lipoprotein (LDL)-receptor activity is responsible for the increase in total cholesterol
and LDL levels observed with age, whereas the generally lower levels in women are mostly due
to the influence of oestrogen (Downer et al., 2014).
Circulating corticosterone levels are widely accepted as biomarkers for stress in vertebrates
(Claunch et al., 2017), and plasma corticosterone levels are commonly used to quantify acute
stress in snakes (Gangloff et al., 2017; Neuman-Lee et al., 2020); however, an elevation in base‐
line blood corticosterone has also been stated to be an indicator of chronic stress in snakes
(van Waeyenberge et al., 2018). There is also the assumption that chronically stressed animals
will show a greater increase in stress hormones when subjected to acute stimuli, such as blood
sampling (Dantzer et al., 2014). The potential elevation of plasma corticosterone levels due to
capture and handling (Herr et al., 2017) brings in a bias that needs to be considered also in
our study, since handling of the snakes for blood sampling was within a time frame of ~5 min
(never exceeding 10 min). Interestingly though, the corticosterone levels determined in our co‐
hort were overall much lower than those reported in wild, free-ranging snakes, such as rat‐
tlesnakes, dice snakes and watersnakes at the time of blood sampling (Lakušić et al., 2020;
Claunch et al., 2017; McCallie and Klukowski, 2022; Lind et al., 2018). This would indicate that
captive snakes, which are at least used to handling, are not significantly stressed by the blood
sampling. However, the interpretation of stress hormone levels is challenging, as they are influ‐
enced by a range of factors, such as age and nutritional status, all well described in mammals
(Reeder and Kramer, 2005), but can also be affected by numerous other, less described factors
in birds and reptiles, including the type of environment, environmental temperature and hu‐
midity, handling and the frequency of feeding (Jaffredo et al., 2006; Holding et al., 2014). Due to
these limitations and the lack of similar studies in wild boa constrictors, it is difficult to com‐
ment to which extent the more ‘standardized’ housing conditions and the frequent handling
by/habituation to humans of the captive snakes in this study may indeed have contributed to
the observed low overall corticosterone and therefore likely low stress levels.
We used fixed and processed buffy coat preparations to gather detailed information on leuko‐
cyte subsets in boa constrictors, following a previously reported approach to the examination
of blood cells in several reptile species (Fontes Pinto et al., 2018). Our results confirm that a
distinction of leukocyte subpopulations is not possible on HE stained sections prepared after
paraffin embedding, confirming that cytological specimens obtained from blood smears are
most suited for this endeavour. However, using immunohistochemistry and RNA-ISH on the
buffy coat preparations, we were able to identify T and B cells for the first time. The results
show that T cells are markedly more numerous than B cells in the peripheral blood of healthy
boas. Since there are so far no studies on lymphocyte subpopulations in reptiles, we rely on the
finding in other animal classes to put this result into context. Indeed, also in birds, i.e. in chick‐
ens, the order phylogenetically closest to reptiles, T cells are far more numerous (> 20%) in the
peripheral blood than B cells (maximally 10%) (Hao et al., 2020). Expression of the T cell
marker CD3 was observed in approximately 12–24% (average) of the lymphoid cells in the
blood of chickens, with a dominance of CD4+T helper cells over CD8+ cytotoxic T cells (Fair et
al., 2008). In the present study, we decided against an attempt to further determine lymphocyte
subsets due to the lack of antibodies for reptiles. More detailed examinations on the
haemolymphatic organs of B.constrictor are now needed to determine the general distribution
of lymphocyte subtypes.
We used a rabbit polyclonal antibody against human Iba1, a calcium binding protein that is up‐
regulated during the activation of macrophages, that we have previously shown to cross react
with snakes (Dervas et al., 2020), to detect monocytes in the boas. Interestingly, the marker ap‐
peared to be expressed also by azurophils (indirect evidence from their frequency and mor‐
phology in the blood smear), which, along with heterophils, thrombocytes and B cells, are con‐
sidered as phagocytic cells in reptiles (Vickie Joseph, 2015, Zimmerman et al., 2010). The litera‐
ture on the classification of monocytes and azurophils in snakes is contradictory. Some authors
state that azurophils should be counted separately in snakes, as their staining properties (e.g.
they are positive in both the PAS and acid phosphatase reaction) render them more similar to
mammalian neutrophils (Zimmerman et al., 2010; Campbell and Grant, 2022). However, be‐
cause their granulopoietic origin has not been confirmed, others claim that monocytes and
azurophils should not be looked upon separately, as the azurophils might represent immature
monocytes (Vickie Joseph, 2015). Similar to other studies, we also found azurophils to be by
far more abundant in the differential blood count (second most common leukocyte subtype)
than monocytes (Nardini et al., 2013; Stacy et al., 2011). It was possible to discern them from
monocytes in the peripheral blood smear as they showed a darker basophilic cytoplasm. The
very limited literature on the ultrastructural features of monocytes and azurophils (Martinez
Silvestre et al., 2005; Salakij et al., 2002), however, did so far not allow direct conclusions on
the appearance of the two cell types in Boa constrictor. The present study sheds light on these,
identifying monocytes as cells with clear cytoplasmic granules, similar to those described in hu‐
man bone marrow promonocytes, blood monocytes and macrophages (Collin et al., 2016).
Azurophils, in contrast, showed intracytoplasmic vacuoles and/or phagocytosed material (indi‐
cating phagocytic activity), but lacked intracytoplasmic granules. Taken together, our findings
suggest that azurophils do not represent an earlier/different developmental stage of a mono‐
cyte but constitute a distinct, fully differentiated, phagocytically active blood cell type in B. con‐
strictor that might indeed originate from the monocytic lineage. Further studies are required to
fully determine the origin and function of azurophils vs monocytes in boas and in snakes in
general, and to determine whether they are still apparent in inflammatory processes.
In summary, the present study provides reference values for relevant haematological and bio‐
chemical markers as well as stress hormone levels in healthy captive B. constrictor, taking into
account age- and sex-related differences. Because data were obtained under ‘standardized’
captive conditions, they should be useful for studies on diseased captive boas and to compare
captive and wild boa populations. The in-depth morphological approach to characterize the
cells of the peripheral blood, using a combined cytologic, histologic, immunohistochemical,
RNA-ISH and ultrastructural approach can pave the way for further research on leukocyte
functions, the composition of haemolymphatic tissues in boa constrictors which have, despite
its large captive global population and distribution, so far not been elucidated. They can also
serve towards studies on immunopathological processes in this species.
Data availability statement
Datasets are available on request: The raw data supporting the conclusions of this article will
be made available by the authors, without undue reservation.
Supplementary Material
Web_Material_coad001
Click here for additional data file.(23K, zip)
Acknowledgements
The authors wish to thank the technical staff of the Histology Laboratory and the Electron
Microscopy Unit of the Institute of Veterinary Pathology, the Veterinary Medical Laboratory
and the Institute for Animal Nutrition and Dietetics, Vetsuisse Faculty, University of Zurich, for
excellent technical support. They are grateful to Dr Jussi Hepojoki, Institute of Veterinary
Pathology, Vetsuisse Faculty, University of Zurich, and Medicum, Department of Virology,
Faculty of Medicine, University of Helsinki, for determination of the CD20 mRNA sequence of
Boa constrictor based on data generated by next generation sequencing.
Contributor Information
E Dervas, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich,

Winterthurerstrasse 268, 8057, Zurich, Switzerland.
E Michalopoulou, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich,

A Liesegang, Institute for Animal Nutrition and Dietetics, Vetsuisse Faculty, University of Zurich,
M Novacco, Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse

260, 8057, Zurich, Switzerland.
F Schwarzenberger, Unit of Physiology, Pathophysiology and Experimental Endocrinology,

Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Veterinä rplatz 1,
1210, Vienna, Austria.
U Hetzel, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich,

A Kipar, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich,

References
Bertona M, Chiaraviglio M (2003) Reproductive biology, mating aggregations, and sexual dimorphism of the Argentine
boa constrictor (Boa constrictor occidentalis). J Herpetol 37: 510–516. 10.1670/122-02A. [CrossRef] [Google
Scholar]
Boes KM, Durham AC, Zachary JF (2017) Bone marrow, blood cells, and the lymphoid/lymphatic system. Pathologic
Basis of Veterinary Disease (JF Zachary, Ed.), Elsevier, Amsterdam, Netherlands. 10.1016/B978-0-323-35775-
3.00013-8. [CrossRef] [Google Scholar]
Brenner D, Lewbart G, Stebbins M, Herman DW (2002) Health survey of wild and captive bog turtles (Clemmys
muhlenbergii) in North Carolina and Virginia. J Zoo Wildl Med 33: 311–316. 10.1638/1042-
7260(2002)033[0311:HSOWAC]2.0.CO;2. [PubMed] [CrossRef] [Google Scholar]
Wahed A, Dasgupta A (2015) Red blood cell disorders. In: Wahed A, Dasgupta A, eds. Hematology and coagulation, 2nd.
Edition. Elsevier, San Diego, pp. 31–53, 10.1016/B978-0-12-800241-4.00003-6. [CrossRef] [Google Scholar]
Campbell TW, Grant KR (2022) Herptile Cytodiagnosis. In Campbell TW (Ed).Exotic Animal Hematology and Cytology,
5th Edition. Wiley-Blackwell, Hoboken NJ, pp. 229–233 [Google Scholar]
Carisch L, Stirn M, Hatt JM, Federer K, Hofmann-Lehmann R, Riond B (2019) White blood cell count in birds: evaluation
of a commercially available method. BMC Vet Res 15: 93. 10.1186/s12917-019-1834-8. [PMC free article] [PubMed]
[CrossRef] [Google Scholar]
Carvalho MPN, Queiroz-Hazarbassanov NGT, Oliveira Massoco C, Sant’Anna SS, Lourenço MM, Levin G, Sogayar MC,
Grego KF, Catão-Dias JL (2017) Functional characterization of neotropical snakes peripheral blood leukocytes
subsets: linking flow cytometry cell features, microscopy images and serum corticosterone levels. Dev Comp
Immunol 74: 144–153. 10.1016/j.dci.2017.04.007. [PubMed] [CrossRef] [Google Scholar]
Casal AB, Camacho M, Ló pez-Jurado LF, Juste C, Oró s J (2009) Comparative study of hematologic and plasma
biochemical variables in eastern Atlantic juvenile and adult nesting loggerhead sea turtles (Caretta caretta). Vet Clin
Pathol 38: 213–218. 10.1111/j.1939-165X.2008.00106.x. [PubMed] [CrossRef] [Google Scholar]
Chiodini RJ, Sundberg JP (1982) Blood chemical values of the common boa constrictor (constrictor constrictor). Am J Vet
Res 43: 1701–1702. [PubMed] [Google Scholar]
Christopher MM, Berry KH, Wallis IR, Nagy KA, Henen BT, Peterson CC (1999) Reference intervals and physiologic
alterations in hematologic and biochemical values of free-ranging desert tortoises in the Mojave Desert. J Wildl Dis
35: 212–238. 10.7589/0090-3558-35.2.212. [PubMed] [CrossRef] [Google Scholar]
Claunch NM, Frazier JA, Escalló n C, Vernasco BJ, Moore IT, Taylor EN (2017) Physiological and behavioral effects of
exogenous corticosterone in a free-ranging ectotherm. Gen Comp Endocrinol 248: 87–96.
10.1016/j.ygcen.2017.02.008. [PubMed] [CrossRef] [Google Scholar]
Collin M, Hughes D, Plü ddemann A, Gordon S (2016) Monocytes, macrophages, and dendritic cells monocytes,
macrophages, and dendritic cells. https://oncohemakey.com/monocytes-macrophages-and-dendritic-cells (last
accessed October 2016).
Cornell A, Gibson KF, Williams TD (2017) Physiological maturity at a critical life-history transition and flight ability at
fledging. Funct Ecol 31: 662–670. 10.1111/1365-2435.12777. [CrossRef] [Google Scholar]
Dantzer B, Fletcher QE, Boonstra R, Sheriff MJ (2014) Measures of physiological stress: a transparent or opaque
window into the status, management and conservation of species? Conserv Physiol 2: cou023.
10.1093/conphys/cou023. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Dervas E, Hepojoki J, Smura T, Prähauser B, Windbichler K, Blü mich S, Ramis A, Hetzel U, Kipar A (2020)
Serpentoviruses: more than respiratory pathogens. J Virol 94: e00649-20. 10.1128/JVI.00649-20. [PMC free article]
[PubMed] [CrossRef] [Google Scholar]
Dickinson VM, Jarchow JL, Trueblood MH (2002) Hematology and plasma biochemistry reference range values for free-
ranging desert tortoises in Arizona. J Wildl Dis 38: 143–153. 10.7589/0090-3558-38.1.143. [PubMed] [CrossRef]
[Google Scholar]
Divers JS (1996) The captive husbandry of captive boa constrictor (Boa constrictor spp.) In: Proceedings of the
Association of Reptilian & Amphibian Veterinarians, pp. 77–81, ARAV (Ed.).
Dolka B, Włodarczyk R, Zbikowski A, Dolka I, Szeleszczuk P, Kluciń ski W (2014) Hematological parameters in relation
to age, sex and biochemical values for mute swans (Cygnus olor). Vet Res Commun 38: 93–100. 10.1007/s11259-014-
9589-y. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Downer B, Estus S, Katsumata Y, Fardo DW (2014) Longitudinal trajectories of cholesterol from midlife through late life
according to apolipoprotein E allele status. Int J Environ Res Public Health 11: 10663–10693.
10.3390/ijerph111010663. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Eklom K, Lill A (2006) Development pattern of blood oxygen carrying capacity in rainbow bee-eater nestlings. Aust J
Zool 54: 1. 10.1071/ZO05063. [CrossRef] [Google Scholar]
El Masri M, Saad AH, Mansour MH, Badir N (1995) Seasonal distribution and hormonal modulation of reptilian T cells.
Immunobiology 193: 15–41. 10.1016/s0171-2985(11)80153-1. [PubMed] [CrossRef] [Google Scholar]
Fair JM, Taylor-McCabe KJ, Shou Y, Marrone BL (2008) Immunophenotyping of chicken peripheral blood lymphocyte
subpopulations: individual variability and repeatability. Vet Immunol Immunopathol 125: 268–273.
10.1016/j.vetimm.2008.05.012. [PubMed] [CrossRef] [Google Scholar]
Fonseca Sarmiento JC, Jaramillo GH, Eduardo Nieto Pico J (2018) Establishment of hematological parameters in boas
(boa constrictor) in the Centro de atencion y valoracion de fauna silvestre del Valle de aburra (Cav amva). Int J Avian
Wildl Biol 3: 146–150. 10.15406/ijawb.2018.03.00076. [CrossRef] [Google Scholar]
Fontes Pinto F, Lopes C, Malhão F, Marcos R (2018) Unraveling avian and reptilian hematology: An optical and electron
microscopic study of the buffy coat. Vet Clin Pathol 47: 407–414. 10.1111/vcp.12640. [PubMed] [CrossRef] [Google
Scholar]
Gangloff EJ, Sparkman AM, Holden KG, Corwin CJ, Topf M, Bronikowski AM (2017) Geographic variation and within-
individual correlations of physiological stress markers in a widespread reptile, the common garter snake
(Thamnophis sirtalis). Comp Biochem Physiol A Mol Integr Physiol 205: 68–76. 10.1016/j.cbpa.2016.12.019. [PubMed]
García-Márquez LJ, Rodríguez-Vázquez A, Leó n-Règagnon V, Osorio-Sarabia D, García-Prieto L (2019) Parasites of boa
constrictor (Squamata: Boidae) captive in Colima, Mexico and their pathological effects. Rex Mex Biodiv 90: e902687.
10.22201/ib.20078706e.2019.90.2687. [CrossRef] [Google Scholar]
Giori L, Stacy NI, Ogle M, Nelson S, Fecteau KA, Cushing AC (2020) Hematology, plasma biochemistry, and hormonal
analysis of captive Louisiana pine snakes (Pituophis ruthveni): effects of intrinsic factors and analytical
methodology. Comp Clin Pathol 29: 145–154. 10.1007/s00580-019-03030-w. [CrossRef] [Google Scholar]
Grego KF, Alves J, Albuquerque LR, Fernandes W (2006) Referências hematoló gicas Para a jararaca de rabo branco
(Bothrops leucurus) recém capturadas da natureza. Arq Bras Med Vet Zootec 58: 1240–1243. 10.1590/S0102-
09352006000600040. [CrossRef] [Google Scholar]
Hao X, Li S, Chen L, Dong M, Wang J, Hu J, Gu M, Wang X, Hu S, Peng Det al. (2020) Establishing a multicolor flow
cytometry to characterize cellular immune response in chickens following H7N9 avian influenza virus infection.
Viruses 12: 1396. 10.3390/v12121396. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Harr KE, Alleman AR, Dennis PM, Maxwell LK, Lock BA, Bennett RA, Jacobson ER (2001) Morphologic and
cytochemical characteristics of blood cells and hematologic and plasma biochemical reference ranges in green
iguanas. J Am Vet Med Assoc 218: 915–921. 10.2460/javma.2001.218.915. [PubMed] [CrossRef] [Google Scholar]
Heo HR, Chen L, An B, Kim KS, Ji J, Hong SH (2015) Hormonal regulation of hematopoietic stem cells and their niche: a
focus on estrogen. Int J Stem Cells 8: 18–23. 10.15283/ijsc.2015.8.1.18. [PMC free article] [PubMed] [CrossRef]
[Google Scholar]
Herr MW, Graham SP, Langkilde T (2017) Stressed snakes strike first: hormone levels and defensive behavior in free
ranging cottonmouths (Agkistrodon piscivorus). Gen Comp Endocrinol 243: 89–95. 10.1016/j.ygcen.2016.11.003.
Hetzel U, Sironen T, Laurinmäki P, Liljeroos L, Patjas A, Henttonen H, Vaheri A, Artelt A, Kipar A, Butcher SJet al. (2013)
Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body
disease. J Virol 87: 10918–10935. 10.1128/JVI.01123-13. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Ho Y-H, Del Toro R, Rivera-Torres J, Rak J, Korn C, García-García A, Macías D, González-Gó mez C, Del Monte A, Wittner
Met al. (2019) Remodeling of bone marrow hematopoietic stem cell niches promotes myeloid cell expansion during
premature or physiological aging. Cell Stem Cell 25: 407–418.e6. 10.1016/j.stem.2019.06.007. [PMC free article]
Holcar M, Goropevš ek A, Ihan A, Avčin T (2015) Age-related differences in percentages of regulatory and effector T
lymphocytes and their subsets in healthy individuals and characteristic STAT1/STAT5 signaling response in helper
T lymphocytes. J Immunol Res 2015: 1–13. 10.1155/2015/352934. [PMC free article] [PubMed] [CrossRef] [Google
Scholar]
Holding ML, Frazier JA, Dorr SW, Pollock NB, Muelleman PJ, Branske A, Henningsen SN, Eikenaar C, Escalló n C,
Montgomery CEet al. (2014) Wet- and dry-season steroid hormone profiles and stress reactivity of an insular dwarf
snake, the Hog Island boa (boa constrictor imperator). Physiol Biochem Zool 87: 363–373. 10.1086/675938.
Hedley J (2022) Boa constrictors, Barrier Animal Care Clinic, Independent Vetcare Ltd, Bristol, UK.
https://www.barriervets.co.uk/exotics/boa-constrictor [Google Scholar]
Arıkan H (2014) Haematology of amphibians and reptiles: a review. Noth-West J Zool 10: 190–209. [Google Scholar]
Hussein MF, Badir N, El Ridi R, Akef M (1979) Lymphoid tissues of the snake, Spalerosophis Diadema, in the different
seasons. Dev Comp Immunol 3: 77–88. 10.1016/S0145-305X(79)80008-7. [PubMed] [CrossRef] [Google Scholar]
Israels JS (2007) Platelet function of the newborn. In: Michelson AD. Platelets, 2nd Edition, Elsevier, Massachusetts,
USA, pp. 431–442, 10.1016/B978-012369367-9/50784-9 [CrossRef] [Google Scholar]
Jaffredo T, Fellah JS, Dunon D (2006) Immunology of Birds and Reptiles. In: Encyclopedia of Life Sciences, John Wiley &
Sons (Ed.), Ltd, London, New York, 10.1038/npg.els.0000521. [CrossRef] [Google Scholar]
Jenkins-Perez J (2012) Hematologic evaluation of reptiles: a diagnostic mainstay. Vet Tec 33: 1–8. [Google Scholar]
Lakuš ić M, Billy G, Bjelica V, Golubović A, Anđelković M, Bonnet X (2020) Effect of capture, phenotype, and
physiological status on blood glucose and plasma corticosterone levels in free-ranging dice snakes. Physiol Biochem
Zool 93: 477–487. 10.1086/711958. [PubMed] [CrossRef] [Google Scholar]
Leceta J, Garrido E, Torroba M, Zapata AG (1989) Ultrastructural changes in the thymus of the turtle Mauremys caspica in
relation to the seasonal cycle. Cell Tissue Res 256: 213–219. 10.1007/BF00224736. [PubMed] [CrossRef] [Google
Scholar]
Leceta J, Zapata A (1985) Seasonal changes in the thymus and spleen of the turtle, Mauremys caspica. A morphometrical,
light microscopical study. Dev Comp Immunol 9: 653–668. 10.1016/0145-305x(85)90030-8. [PubMed] [CrossRef]
[Google Scholar]
Lee SH, Kim JW, Lee BC, Oh HJ (2020) Age-specific variations in hematological and biochemical parameters in middle-
and large-sized of dogs. J Vet Sci 21: e7. 10.4142/jvs.2020.21.e7. [PMC free article] [PubMed] [CrossRef] [Google
Scholar]
Lind C, Moore IT, Akçay Ç, Vernasco BJ, Lorch JM, Farrell TM (2018) Patterns of circulating corticosterone in a
population of rattlesnakes afflicted with Snake fungal disease: stress hormones as a potential mediator of seasonal
cycles in disease severity and outcomes. Physiol Biochem Zool 91: 765–775. 10.1086/695747. [PubMed] [CrossRef]
[Google Scholar]
Lisičić D, Đikić D, Benković V, Horvat Knež ević A, Orš olić N, Tadić Z (2013) Biochemical and hematological profiles of
a wild population of the nose-horned viper Vipera ammodytes (Serpentes: viperidae) during autumn, with a
morphological assessment of blood cells. Zool Stud 52: 11. 10.1186/1810-522X-52-11. [CrossRef] [Google Scholar]
Machado CC, Silva LFN, Ramos PRR, Takahira RK (2006) Seasonal influence on hematologic values and hemoglobin
electrophoresis in Brazilian boa constrictor amarali. J Zoo Wildl Med 37: 487–491. 10.1638/05-124.1. [PubMed]
Martinez Silvestre A, Marco I, Domínguez MÁ , Lavin S, Cuenca R (2005) Morphology, cytochemical staining, and
ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi). Res Vet Sci 78:
127–134. 10.1016/j.rvsc.2004.07.009. [PubMed] [CrossRef] [Google Scholar]
McCallie KL, Klukowski M (2022) Corticosterone in three species of free-ranging watersnakes: testing for reproductive
suppression and an association with body condition. Comp Biochem Physiol A Mol Integr Physiol 269: 111214.
10.1016/j.cbpa.2022.111214. [PubMed] [CrossRef] [Google Scholar]
McMillan DB, Harris RJ (Eds.)(2018) Blood and Lymph. In: An Atlas of Comparative Vertebrate Histology, Elsevier,
United Kingdom, London, pp.171–201, 10.1016/B978-0-12-410424-2.00007-X. [CrossRef] [Google Scholar]
Mehta S, Sinha R (2018) Comparative transmission electron microscopic studies on monocytes of horse and dog.
Haryana Veterinarian 58: 205–206. [Google Scholar]
Moestl E, Maggs JL, Schrö tter G, Besenfelder U, Palme R (2002) Measurement of cortisol metabolites in faeces of
ruminants. Vet Res Commun 26: 127–139. 10.1023/a:1014095618125. [PubMed] [CrossRef] [Google Scholar]
Moller CA, Gaál T, Mills JN (2016) The hematology of captive bobtail lizards (Tiliqua rugosa): blood counts, light
microscopy, cytochemistry, and ultrastructure. Vet Clin Pathol 45: 634–647. 10.1111/vcp.12425. [PubMed]
Montgomery CE, Boback SM, Reed RN, Frazier JA (eds) (2015) An assessment of the impact of the pet trade on five CITES-
Appendix II case studies - Boa constrictor imperator. International Union for the Conservation of Nature, Honduras
[Google Scholar]
Morgan KN, Tromborg CT (2007) Sources of stress in captivity. Appl Anim Behav Sci 102: 262–302.
10.1016/j.applanim.2006.05.032. [CrossRef] [Google Scholar]
Morpurgo B, Gelman A (1991) The effect of age, sex and diet on the plasma cholesterol levels of the Nile crocodile,
Crocodylus niloticus. Comp Biochem Physiol A Mol Integr Physiol 99: 687–689. 10.1016/0300-9629(91)90151-2.
Myers P, Espinosa R, Parr CD, Jones T, Hammond GS, Dewey TA (2022) The Animal Diversity Web (online), Michigan,
USA. https://animaldiversity.org
Nardini G, Leopardi S, Bielli M (2013) Clinical Hematology in reptilian species. Vet Clin North Am Exot Anim Pract 16:
1–30. 10.1016/j.cvex.2012.09.001. [PubMed] [CrossRef] [Google Scholar]
Neuman-Lee LA, Hudson SB, Webb AC, French SS (2019) Investigating the relationship between corticosterone and
glucose in a reptile. J Exp Biol 223: jeb203885. 10.1242/jeb.203885. [PubMed] [CrossRef] [Google Scholar]
Pereira MA, Alexandre-Pires G, Câmara M, Santos M, Martins C, Rodrigues A, Adriana J, Passero LFD, Da Pereira FI,
Santos-Gomes G (2019) Canine neutrophils cooperate with macrophages in the early stages of Leishmania infantum
in vitro infection. Parasite Immunol 41: e12617. 10.1111/pim.12617. [PubMed] [CrossRef] [Google Scholar]
Pierezan F, Mansell J, Ambrus A, Hoffmann AR (2014) Immunohistochemical expression of ionized calcium binding
adapter molecule 1 in cutaneous histiocytic proliferative, neoplastic and inflammatory disorders of dogs and cats. J
Comp Pathol 151: 347–351. 10.1016/j.jcpa.2014.07.003. [PubMed] [CrossRef] [Google Scholar]
Piewbang C, Wardhani SW, Poonsin P, Yostawonkul J, Chai-in P, Lacharoje S, Saengdet T, Vasaruchapong T,

Boonrungsiman S, Kongmakee Pet al. (2021) Epizootic reptilian ferlavirus infection in individual and multiple
snake colonies with additional evidence of the virus in the male genital tract. Sci Rep 11: 12731. 10.1038/s41598-
021-92156-5. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Quadrini AE, Garcia VC, Freire BC, Martins M (2018) Haematological reference of snakes: Amazon tree boa (Corallus
hortulanus, Linnaeus, 1758) and Burmenese python (Python bivittatus, Kuhl, 1820) in captive. Arq Bras Med Vet
Zootec 70: 1172–1178. 10.1590/1678-4162-9865. [CrossRef] [Google Scholar]
Raskin RE (Ed.), Latimer KS, Tvedten H (Ed.) (2004) Leukocyte disorders. Small Animal Clinical Diagnosis by Laboratory
Methods, Elsevier, Amsterdam, Netherlands, pp. 63–91. 10.1016/B0-72-168903-5/50008-2. [CrossRef] [Google
Scholar]
Reeder DM, Kramer KM (2005) Stress in free-ranging mammals: integrating physiology, ecology, and natural history. J
Mammal 86: 225–235. 10.1644/BHE-003.1. [CrossRef] [Google Scholar]
Rousselet E, Stacy NI, LaVictoire K, Higgins BM, Tocidlowski ME, Flanagan JP, Godard-Codding CA (2013) Hematology
and plasma biochemistry analytes in five age groups of immature, captive-reared Loggerhead Sea turtles (Caretta
caretta). J Zoo Wildl Med 44: 859–874. 10.1638/2012-0162R1.1. [PubMed] [CrossRef] [Google Scholar]
Salakij C, Salakij J, Suthunmapinunta P, Chanhome L (2002) Hematology, morphology and ultrastructure of blood cells
and blood parasites from puff-faced watersnakes (Homalopsis buccata). Kasetsart J 36: 35–43. [Google Scholar]
Schumacher J (2006) Selected infectious diseases of wild reptiles and amphibians. J Exot Pet Med 15: 18–24.
10.1053/j.jepm.2005.11.004. [CrossRef] [Google Scholar]
Sealander JA (1964) The influence of body size, season, sex, age and other factors upon some blood parameters in small
mammals. J Mammal 45: 598. 10.2307/1377331. [CrossRef] [Google Scholar]
Silva LF, Riani-Costa CC, Ramos PR, Takahira R (2011) Seasonal influence on biochemical profile and serum protein
electrophoresis for Boa constrictor amarali in captivity. Braz J Biol 71: 517–520. 10.1590/S1519-
69842011000300023. [PubMed] [CrossRef] [Google Scholar]
Stacy NI, Alleman AR, Sayler KA (2011) Diagnostic hematology of reptiles. Clin Lab Med 31: 87–108.
10.1016/j.cll.2010.10.006. [PubMed] [CrossRef] [Google Scholar]
Svoboda M, Knotek Z, Knotkova Z, Trnkova S (eds) (2006) Advances in reptilian hematology and blood chemistry. In
31st World Small Animal Veterinary Association World Congress, Elsevier, Prague, Czech Republic, 11–14 Oct [Google
Scholar]
Trimboli F, Amicis I, Di Loria A, Ceniti C, Carluccio A (2020) Reference ranges for hematological and biochemical
profile of Martina franca donkeys. Front vet sci 7: 602984. 10.3389/fvets.2020.602984. [PMC free article] [PubMed]
Vácha J, Znojil V (1981) The allometric dependence of the life span of erythrocytes on body weight in mammals. Comp
Biochem Physiol A 69: 357–362. 10.1016/0300-9629(81)92990-X. [CrossRef] [Google Scholar]
Van Waeyenberge J, Aerts J, Hellebuyck T, Pasmans F, Martel A (2018) Stress in wild and captive snakes: quantification,
effects and the importance of management. VDT 87: 59–65. 10.21825/vdt.v87i2.16082. [CrossRef] [Google Scholar]
Joseph V (2015) Reptile clinical pathology. In ExoticsCon, San Antonio, Texas, USA, 29 Aug-2 Sep [Google Scholar]
Wahed A, Dasgupta A (2015b) Red blood cell disorders. In: Wahed A, Dasgupta A, eds. Hematology and coagulation, 2nd.
Edition. Elsevier, San Diego, pp. 31–53, 10.1016/B978-0-12-800241-4.00003-6. [CrossRef] [Google Scholar]
Wang D, Tanaka-Yano M, Meader E, Kinney MA, Morris V, Da Lummertz RE, Liu N, Liu T, Zhu Q, Orkin SHet al. (2022)
Developmental maturation of the hematopoietic system controlled by a Lin28b-let-7-Cbx2 axis. Cell Rep 39: 110587.
10.1016/j.celrep.2022.110587. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Warwick C, Arena P, Lindley S, Jessop M, Steedman C (2013) Assessing reptile welfare using behavioural criteria. In
Pract 35: 123–131. 10.1136/inp.f1197. [CrossRef] [Google Scholar]
Wood RD (2014) Leukogram Abnormalities, Merck & Co., Inc., Rahway, NJ, USA. https://www.msdvetmanual.com (last
accessed March 2022). [Google Scholar]
Zimmerman LM, Vogel LA, Edwards KA, Bowden RM (2010) Phagocytic B cells in a reptile. Biol Lett 6 (2): 270–273.
10.1098/rsbl.2009.0692. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

Haematology, Biochemistry and Morphological Features of Peripheral Blood Cells in Captive Boa Constrictor - PMC

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Haematology, Biochemistry and Morphological Features of Peripheral Blood Cells in Captive Boa Constrictor - PMC

Uploaded by

Copyright:

Available Formats

20/10/23, 9:47 Haematology, biochemistry and morphological features of peripheral blood cells in captive Boa constrictor - PMC

Conserv Physiol. 2023; 11(1): coad001. PMCID: PMC9885740

Haematology, biochemistry and morphological features of peripheral blood cells in

Steven Cooke, Editor

Although knowledge on appropriate maintenance and upbringing of captive snakes is growing,

Material and Methods

Buffy coat preparation, processing and staining, immunohistochemistry, RNA-ISH and

Morphological identification of blood cells

Haematological parameters (total leukocyte count, haematocrit, haemoglobin, mean cell

Animals and disease/infection state

Cell type Female Male Test Total

<3 ≥3 Total <3 ≥3 Total para

years years years years meters

(n) (n) (n) (n) (n) (n) (n)

Leukocytes (3) (16) (20) (6) (11) (17) t= (49)

t = 0.4162, df = 17, P t = −0.1660, df = 15,

Lymphocytes (3) (16) (20) (6) (11) (17) t= (49)

t = 1.1424, df = 17, P t = 0.8873, df = 15, P

Heterophils (3) (16) (20) (6) (11) (17) z= (49)

Eosinophils (3) (10) (14) (3) (7) (10) t= (34)

**Lower (upper) confidence limit held at minimum (maximum) of sample.

Relative leukocyte percentages of clinically healthy captive B. constrictor.

Cell type Female Male Total

<3 ≥3 Total <3 ≥3 Total

(n) (n) (n) (n) (n) (n) Test (n)

Lymphocytes (3) (16) (20) (6) (11) (17) t= (49)

t = 2.4938, df = 17, P t = 2.3456, df = 15, P

t = −1.6721, df = 17, t = −1.2361, df = 15,

Heterophils (3) (16) (20) (6) (11) (17) t= (49)

t = −1.8303, df = 17, t = −1.7953, df = 15,

Eosinophils (3) (10) (14) (3) (7) (10) t= (34)

t = −0.1540, df = 11, t = 1.8867, df = 8, P =

Monocytes (2) (2) (15) (18) (6) (11) (17) t= (47)

Haematocrit (3) (14) (18) (6) (8) (14) t= (44)

t = −1.4892, df = 15, P t = −0.7739, df = 12,

Haemoglobin (3) 7.033 (16) (20) (6) (11) (17) t= (49)

t = −1.4631, df = 17, P t = −1.4830, df = 17,

MCHC (1) (3) I16) (20) (6) (11) (17) t= (49)

t = 0.0452, df = 17, P t = −1.0662, df = 15,

<3 years ≥3 years

Female Male Total Female Male Total

Haematocrit (3) (6) (21) (14) (8) (22) t= (44)

t = 1.7413, df = 7, P = t = 1.9316, df = 20, P

Age- and sex-associated differences in blood parameters of healthy B. constrictor. A, B. Distribution of

Identification and morphological features of blood cells

Lymphocytes In the cytological specimens, lymphocytes presented as small- to medium-sized

Biochemical parameters and corticosteroid levels

Biochemical parameters and corticosterone levels of clinically healthy captive B. constrictor.

Para-meter <3 ≥3 Total <3 ≥3 Total Test Total

(n) (n) (n) (n) (n) (n) (n)

Total protein (5) (20) (25) (7) (19) (26) t= (66)

t = −2.4273, df = 23, t = −1.6696, df = 24,

Albumin (1) (5) (20) (25) (7) (19) (26) t= (66)

t = −2.4591, df = 23, t = −1.8487, df = 24,

LDH (2) (5) (20) (25) (7) (19) (26) t= (66)

t = −1.2396, df = 23, t = 0.4138, df = 24, P

AST (2) (5) (20) (25) (7) (19) (26) t= (66)

t = 0.2900, df = 23, P t = 0.0999, df = 24, P

Cholesterol (2) (5) (20) (25) (7) (19) (26) t= (66)

** Lower (upper) confidence limit held at minimum (maximum) of sample

In contrast to the lymphocyte percentages, we observed an increase in the percentage of het‐

Data availability statement

Click here for additional data file.(23K, zip)

E Dervas, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich,